Bottom Line:
Using this approach, InsP6, InsP7 and InsP8 were visualized in Dictyostelium extracts and a variety of mammalian cell lines and tissues, and the effects of metabolic perturbation on these were explored.Firstly, there is an active InsP6 phosphatase in human plasma, and secondly, InsP6 is undetectable in either fluid.These observations seriously question reports that InsP6 is present in human biofluids and the advisability of using InsP6 as a dietary supplement.

Affiliation: Medical Research Council Laboratory for Molecular Cell Biology, University College London, London, UK.

ABSTRACTInositol phosphates are a large and diverse family of signalling molecules. While genetic studies have discovered important functions for them, the biochemistry behind these roles is often not fully characterized. A key obstacle in inositol phosphate research in mammalian cells has been the lack of straightforward techniques for their purification and analysis. Here we describe the ability of titanium dioxide (TiO2) beads to bind inositol phosphates. This discovery allowed the development of a new purification protocol that, coupled with gel analysis, permitted easy identification and quantification of InsP6 (phytate), its pyrophosphate derivatives InsP7 and InsP8, and the nucleotides ATP and GTP from cell or tissue extracts. Using this approach, InsP6, InsP7 and InsP8 were visualized in Dictyostelium extracts and a variety of mammalian cell lines and tissues, and the effects of metabolic perturbation on these were explored. TiO2 bead purification also enabled us to quantify InsP6 in human plasma and urine, which led to two distinct but related observations. Firstly, there is an active InsP6 phosphatase in human plasma, and secondly, InsP6 is undetectable in either fluid. These observations seriously question reports that InsP6 is present in human biofluids and the advisability of using InsP6 as a dietary supplement.

RSOB150014F1: TiO2 purifies inositol phosphates. (a)Flowchart describing the five-step TiO2 bead extractionprocedure. (i) Acidic solution (blue) containing inositol phosphatesis incubated with (ii) TiO2 beads (yellow) for 10 min,before (iii) spinning and washing the beads twice with 1 M PA.Elution occurs by incubation (iv) at basic pH (red) with subsequentspinning and recovering the supernatant (v). This is evaporated (vi)to concentrate and neutralize (grey) the extract.(b) InsP6 diluted in 1 M PA waspurified using TiO2 and subjected to PAGE with toluidineblue staining. I, input; S, supernatant; W1 and W2, washes; E,eluted. While all the eluted InsP6 was loaded on the gelonly 1/10 (approx. 100 µl) of the S, W1 and W2 fractions wereloaded. The acid in these fractions results in slightly compressedand slower migration of the orange G dye. (c) As(b), but using a PA extraction from vegetativestate D. discoideum cells as input. These toluidineblue-stained gels are representative of experiments performed atleast three times. (d) To calculate the exactpercentage of recovery, radioactive3H-Ins(1,4,5)P3 and3H-InsP6 were subjected to TiO2purification. The radioactivity recovered (E) and radioactivityremaining on TiO2 beads (B) were normalized to therespective radioactive input (I). The graph showing the average± s.d. (n = 4) is representative oftwo independent experiments with matching results.

Mentions:
The ability of titanium dioxide (TiO2) to bind with very high affinityto phosphate groups is used in phosphopeptide enrichment protocols, an essentialstep in modern phosphoproteomic studies [24]. We used this TiO2 property [25] to develop a simpleenrichment method (schematized in figure1a) to extract inositol phosphates from acidicsolutions, normally 1 M PA [19]. Initially, a specific amount of InsP6 diluted in PA wasincubated with TiO2 beads for 30 min. After two washes with PA,InsP6 was eluted from the beads by a pH change induced by10% ammonium hydroxide. After removing the ammonium hydroxide andreducing the volume using a centrifugal evaporator, the samples were resolved byPAGE and visualized with toluidine blue staining, demonstrating an almostcomplete recovery of the input InsP6 (figure 1b). We next tested thisprocedure on a D. discoideum extract, and recoveredquantifiable levels of InsP6 and its pyrophosphate derivativesInsP7 and InsP8 (figure 1c). To precisely quantifythe inositol phosphate recovery, radioactive3H-Ins(1,4,5)P3 and 3H-InsP6tracers were each mixed with 2 nmol of Ins(1,4,5)P3 andInsP6 and subjected to TiO2 enrichment. Theseexperiments demonstrated that 87 ± 4.6 and 84 ± 3.5%(average ± s.d., n = 4) forIns(1,4,5)P3 and InsP6, respectively, of inputradioactivity was recovered after TiO2 elution, while about2–4% remained attached to the TiO2 beads (figure 1d). TheTiO2 beads are in fact completely efficient at binding andreleasing inositol phosphates; the small loss is intrinsic to the manualhandling involved. Figure 1.

RSOB150014F1: TiO2 purifies inositol phosphates. (a)Flowchart describing the five-step TiO2 bead extractionprocedure. (i) Acidic solution (blue) containing inositol phosphatesis incubated with (ii) TiO2 beads (yellow) for 10 min,before (iii) spinning and washing the beads twice with 1 M PA.Elution occurs by incubation (iv) at basic pH (red) with subsequentspinning and recovering the supernatant (v). This is evaporated (vi)to concentrate and neutralize (grey) the extract.(b) InsP6 diluted in 1 M PA waspurified using TiO2 and subjected to PAGE with toluidineblue staining. I, input; S, supernatant; W1 and W2, washes; E,eluted. While all the eluted InsP6 was loaded on the gelonly 1/10 (approx. 100 µl) of the S, W1 and W2 fractions wereloaded. The acid in these fractions results in slightly compressedand slower migration of the orange G dye. (c) As(b), but using a PA extraction from vegetativestate D. discoideum cells as input. These toluidineblue-stained gels are representative of experiments performed atleast three times. (d) To calculate the exactpercentage of recovery, radioactive3H-Ins(1,4,5)P3 and3H-InsP6 were subjected to TiO2purification. The radioactivity recovered (E) and radioactivityremaining on TiO2 beads (B) were normalized to therespective radioactive input (I). The graph showing the average± s.d. (n = 4) is representative oftwo independent experiments with matching results.

Mentions:
The ability of titanium dioxide (TiO2) to bind with very high affinityto phosphate groups is used in phosphopeptide enrichment protocols, an essentialstep in modern phosphoproteomic studies [24]. We used this TiO2 property [25] to develop a simpleenrichment method (schematized in figure1a) to extract inositol phosphates from acidicsolutions, normally 1 M PA [19]. Initially, a specific amount of InsP6 diluted in PA wasincubated with TiO2 beads for 30 min. After two washes with PA,InsP6 was eluted from the beads by a pH change induced by10% ammonium hydroxide. After removing the ammonium hydroxide andreducing the volume using a centrifugal evaporator, the samples were resolved byPAGE and visualized with toluidine blue staining, demonstrating an almostcomplete recovery of the input InsP6 (figure 1b). We next tested thisprocedure on a D. discoideum extract, and recoveredquantifiable levels of InsP6 and its pyrophosphate derivativesInsP7 and InsP8 (figure 1c). To precisely quantifythe inositol phosphate recovery, radioactive3H-Ins(1,4,5)P3 and 3H-InsP6tracers were each mixed with 2 nmol of Ins(1,4,5)P3 andInsP6 and subjected to TiO2 enrichment. Theseexperiments demonstrated that 87 ± 4.6 and 84 ± 3.5%(average ± s.d., n = 4) forIns(1,4,5)P3 and InsP6, respectively, of inputradioactivity was recovered after TiO2 elution, while about2–4% remained attached to the TiO2 beads (figure 1d). TheTiO2 beads are in fact completely efficient at binding andreleasing inositol phosphates; the small loss is intrinsic to the manualhandling involved. Figure 1.

Bottom Line:
Using this approach, InsP6, InsP7 and InsP8 were visualized in Dictyostelium extracts and a variety of mammalian cell lines and tissues, and the effects of metabolic perturbation on these were explored.Firstly, there is an active InsP6 phosphatase in human plasma, and secondly, InsP6 is undetectable in either fluid.These observations seriously question reports that InsP6 is present in human biofluids and the advisability of using InsP6 as a dietary supplement.

Affiliation:
Medical Research Council Laboratory for Molecular Cell Biology, University College London, London, UK.

ABSTRACTInositol phosphates are a large and diverse family of signalling molecules. While genetic studies have discovered important functions for them, the biochemistry behind these roles is often not fully characterized. A key obstacle in inositol phosphate research in mammalian cells has been the lack of straightforward techniques for their purification and analysis. Here we describe the ability of titanium dioxide (TiO2) beads to bind inositol phosphates. This discovery allowed the development of a new purification protocol that, coupled with gel analysis, permitted easy identification and quantification of InsP6 (phytate), its pyrophosphate derivatives InsP7 and InsP8, and the nucleotides ATP and GTP from cell or tissue extracts. Using this approach, InsP6, InsP7 and InsP8 were visualized in Dictyostelium extracts and a variety of mammalian cell lines and tissues, and the effects of metabolic perturbation on these were explored. TiO2 bead purification also enabled us to quantify InsP6 in human plasma and urine, which led to two distinct but related observations. Firstly, there is an active InsP6 phosphatase in human plasma, and secondly, InsP6 is undetectable in either fluid. These observations seriously question reports that InsP6 is present in human biofluids and the advisability of using InsP6 as a dietary supplement.